Neurofilaments (NF), the intermediate filaments (IF) specific for neurons, are an assembly of three subunits of apparent Mr on SDS-PAGE of 68 kD, 160 kD and 200 kD, termed NF-L, NF-M and NF-H, respectively. All three subunits contain a highly conserved helical rod domain. The two heavier subunits also have extended C-terminal tail domains which are heavily phosphorylated. The cDNA-derived sequences of the two heavy NF-subunits have revealed the presence of 5, 12 and 40 Lys-Ser-Pro (Val,Ala,X) repeats in the C-terminal domains of rat NF-M, human NF-M and human NF-H, respectively (Napolitano et al., 1987; Myers et al., 1987 and Lees et al., 1988). These sequences form the epitopes of several phosphoepitope-specific anti-NF-mAbs (Lee et al., 1988). The physiological significance of NF and their phosphorylation is not very well understood yet (reviewed by Matus, 1988); correlative evidence suggests involvement in the regulation of axonal diameter (Hoffman et al., 1987; Pleasure et al., 1989). Electron microscopic studies in conjunction with antibody decoration (Hirokawa wt al., 1984) and biochemical evidence (Minami et al., 1983) favor NF-H as a component in interactions of the NF and microtubule networks. The phosphorylation status of NF and their ability to promote tubulin polymerization are correlated in vitro (Minami et al., 1985).
The existence of NF-kinase(s) not activated by common second messengers and some of their expected properties were postulated from in vivo phosphorylation studies on extruded axoplasm of the giant axons of the squid (Pant et al., 1978, 1986) and of Myxicola (Shecket et al., 1982). In vitro characterization of purified NF-kinases has focused so far on activities that copurify with the NF-cytoskeleton and can be dissociated under high salt conditions (Runge et al., 1981; Toru-Delbauffe et al., 1983). There is currently no evidence of second messenger dependence of any of these activities. From a mixture of such kinases one 67 kD activity has been purified to apparent homogeneity (Wible et al., 1989). This kinase prefers NF-H as a substrate, but only if not completely dephosphorylated. A cAMP-dependent kinase copurifying with microtubules has been shown to phosphorylate preferentially NF-M in NF-triplets (Leterrier et al., 1981). In no case are the stoichiometry or the sites of phosphorylation known and no shift of apparent Mr of NF-M and NF-H on SDS-PAGE has been demonstrated. Such a shift is expected after incorporation of phosphate in high stoichiometric ratios into the dephosphorylated subunits. A smaller than expected gel shift associated with a heterogeneous state of KSP-phosphorylation of NF-M is induced by uncharacterized kinases in mouse L cells transfected with a human NF-M clone (Pleasure et al., 1990).
A possible pathological role of aberrant NF-phosphorylation was considered when the anti-rat-NF mAb 07-5 (commercially available as SMI-34 from Sternberger-Meyer Immunochemicals of Jarretsville, Md., U.S.A.) was found to stain neurofibrillary tangles in brain tissue from Alzheimer's patients (Sternberger et al., 1985), but did not stain normal human brain tissue, except for cerebellar basket cell axons and certain motoneuron axons of patients 60 years of age (Blanchard & Ingram, 1989). On the other hand, there is a report that the localization of the SMI-34 epitope is exclusively perikaryonal, while most other mAbs reacting with NF-phosphoepitopes stain axons preferentially (Sternberger et al., 1983).
However, immunochemical evidence (Grundke-lqbal et al., 1986; Kosik et al., 1986; Wood et al., 1986; Nukina et al., 1987) concerning the crossreactivities of a series of mAbs with NFs, microtubule associated protein TAU and the main component of tangles and paired helical filaments (PHF) point to TAU as a major constituent of PHFs. This deduction is reinforced by the isolation from PHFs of TAU-derived peptides (Wischik et al,, 1988), while no NF-derived peptides (Kondo et al., 1988) were obtained. A number of anti-NF mAbs crossreacting with TAU, among them SMI-31 (commercially available from Sternberger-Meyer Immunochemical) and RT97, recognize the phosphorylated KSP-sequence repeat in NF proteins (Lee et al., 1988). PHFs react strongly with RT97, but only after prolonged treatment with SDS, suggesting the presence of this phosphorylated epitope in PHF in a nonperipheral location (Rasool et al., 1984). Several lines of evidence indicate an abnormal level or an abnormal site of phosphorylation in the C-terminal portion of the TAU molecule in Alzheimer's Disease (AD) (Grundke-lqbal et al., 1986; Kondo et al., 1988; Iqbal et al., 1989). If an abnormally phosphorylated form of TAU is responsible for or involved in the development of neurological conditions characterized by PHFs in neurofibrillary tangles, then it clearly would be extremely important to identify the factor(s) which cause that phosphorylation.